Project Abstract Ferroptosis is a non-apoptotic, oxidative, iron-dependent form of cell death that can be triggered in cancer and other cells. Ferroptosis can be induced by depriving cells of the amino acid cysteine, a precursor to the essential intracellular antioxidant glutathione. A major gap in our knowledge concerns the molecular mechanisms governing cancer cell susceptibility to ferroptosis. Our preliminary data suggest that stabilization of wild-type p53 and transcription of the canonical p53 target gene CDKN1A (encoding p21) suppresses ferroptosis through a mechanism involving the conservation of intracellular GSH and suppression of toxic lipid reactive oxygen species (ROS). This proposal will test the hypothesis that the p53-p21 pathway suppresses ferroptosis by inhibiting cyclin-dependent kinase (CDK)-mediated phosphorylation of key enzymes involved in ROS metabolism. Biochemical, genetic, and proteomic approaches will be used to explore the link between p21, CDKs, and ROS metabolism. Cell models and chemical techniques will be used to validate the generalizability of this phenotype to diverse cancer cell types and to cell culture systems mimicking physiological stresses encountered by tumors in vivo. These studies will be performed in human and mouse cancer cell lines and aided by high-throughput time-lapse cell death imaging. This research will provide a link between the p53-pathway, non-apoptotic cell death, and metabolism, and may inform therapies designed to selectively induce ferroptosis in cancer cells. !